EP2019145A1 - Transduction de gène de cellule souche hématopoïétique - Google Patents

Transduction de gène de cellule souche hématopoïétique Download PDF

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EP2019145A1
EP2019145A1 EP08018067A EP08018067A EP2019145A1 EP 2019145 A1 EP2019145 A1 EP 2019145A1 EP 08018067 A EP08018067 A EP 08018067A EP 08018067 A EP08018067 A EP 08018067A EP 2019145 A1 EP2019145 A1 EP 2019145A1
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cells
human
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hematopoietic progenitor
stem cells
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Stanton L. Gerson
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Case Western Reserve University
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Definitions

  • the present invention relates to the genetic transformation of cells and more particularly to the transformation of hematopoietic stem cells.
  • the present invention is directed to human hematopoietic stem cells that are genetically engineered to carry within them genes of interest, particularly for the expression of physiologically or pharmacologically active proteins.
  • human mesenchymal stem cells can be used to enhance the transduction of human hematopoietic progenitor cells for the expression of exogenous gene products.
  • the mesenchymal stem cells can be allogeneic or autologous to the hematopoietic progenitor cells.
  • the human hematopoietic progenitor cells are CD34+ cells.
  • mesenchymal stem cells as described herein provides a method of enhancing gene transduction with a reagent that may be derived from alternate sources in that either autologous or allogeneic mesenchymal stem cells may be used for the method described herein.
  • the mesenchymal stem cells represent a well characterized cell population which can be prepared in a much more reproducible manner than, for example, the heterogeneous Dexter-like stromal culture.
  • Dexter stroma in addition to MSCs, contains T and B lymphocytes, macrophages, dendritic cells and endothelial cells.
  • the mesenchymal stem cells may be used in the absence or presence of exogenous growth factors or cytokines.
  • the invention relates to a method of transducing human hematopoietic stem cells to express an incorporated genetic material of interest.
  • Human hematopoietic stem cells are isolated from an individual donor.
  • the isolated hematopoietic progenitor cells are transduced in the presence of human mesenchymal stem cells such that they are able to express exogenous gene products. It is contemplated that the transformed cells and the expression products of the incorporated genetic material can be used alone or in combination with other cells and/or compositions.
  • the transduced human hematopoietic progenitor cells are separated from the human mesenchymal stem cells prior to utilization of the transduced hematopoietic stem cells for their intended purpose.
  • the invention involves a method of modifying human hematopoietic progenitor cells with genetic material of interest in the presence of the mesenchymal stem cells.
  • the transduced hematopoietic stem cells genes may be used to produce the material of interest, for example, a protein or polypeptide, by in vitro expression of the protein which may be useful for specific therapeutic applications or non-therapeutic applications.
  • the genetic material of interest may express, for example, intracellular gene products, signal transduction molecules, cell surface proteins, extracellular gene expression products and hormone receptors.
  • the hematopoietic stem cells are transduced ex-vivo for subsequent in vivo use, i.e. transduced with a polynucleotide encoding a therapeutic protein and the cells administered to a mammal in need thereof for in vivo expression of a therapeutically effective amount.
  • Examples of genetic material for transduction into hematopoietic progenitor cells includes the expression of gene products which have a role in hematopoietic cell maintenance, tissue development, remodeling, repair and in vivo production of extracellular gene products.
  • Fig. 1 Drug resistance in hematopoietic progenitors transduced in the presence of hMSCs. Shown is a comparison of the number of colonies of cells, transduced with ⁇ MGMT in the presence of hMSCs ( Fig. 1A ); fibronectin (FN) ( Fig. 1B ), and Dexter stroma ( Fig. 1C ) surviving after exposure of the transduced cells to TMZ or BCNU, as compared to untransduced cells under the same conditions. Error bars represent SEM.
  • FIG. 2 AGT expression in simultaneously transduced hMSCs and CD34+ cells.
  • CD34+ cells and hMSCs were cultured in the presence of retroviral supernatant for 72 hours.
  • CD34+ cells were plated for 5 hours to deplete adherent cells while hMSCs were trypsinized and further cultured for 1 week with media changes.
  • AGT expression was determined in both cell types by flow cytometry using the monoclonal antibody mT3.1. Shown is the per cent AGT expression in transduced cells versus nontransduced cells in hematopoietic cells ( Fig. 2A ) and hMSCs ( Fig. 2B ).
  • the present invention provides a method of transducing hematopoetic progenitor cells comprising contacting the hematopoietic progenitor cells with exogenous genetic material and human mesenchymal stem cells.
  • the mesenchymal stem cells are autologous to the hematopoietic progenitor cells. In another aspect, the mesenchymal stem cells are allogeneic to the hematopoietic progenitor cells.
  • the transduced human hematopoietic progenitor cells and the mesenchymal stem cells may be used together.
  • the human hematopoietic progenitor cells are separated from the mesenchymal stem cells after the hematopoietic progenitor cells have been transduced.
  • One of skill in the art would be able to ascertain which mode of use would be suitable depending on the purpose for the transduced cells.
  • Isolated human hematopoietic stem cells have been described, for example, in Tsuksamoto et al., U.S. Patent No. 5,061,620 (1991 ) and reviewed in Edgington, Biotechnology, 10:1099-1106 (1992 ) and the references cited therein. These are distinguished from mesenchymal stem cells by their ability to differentiate into myeloid and lymphoid blood cells.
  • the hematopoietic progenitor cells can be obtained according to known methods, for example, from peripheral blood mononuclear cells or bone marrow.
  • the human hematopoietic progenitor cells are CD34+ cells. These progenitor hematopoietic cells can be isolated, for example, by positive antibody selection.
  • MSCs Mesenchymal stem cells
  • the mesenchymal stem cells can be isolated and prepared according to methods known in the art, for example, a process for isolating, purifying, and expanding the marrow-derived mesenchymal stems cells in culture, i.e. in vitro , is described in U.S. Patent Nos. 5,197,985 and 5,226,914 and PCT Publication No. WO 92/22584 (1992 ) as well as numerous literature references by Caplan and Haynesworth.
  • the stem cells may be isolated from other cells by density gradient fractionation, such as by Percoll gradient fractionation.
  • the human mesenchymal stem cells also can include a cell surface epitope specifically bound by antibodies from hybridoma cell line SH2, deposited with the ATCC under accession number HB 10743; antibodies from hybridoma cell line SH3, deposited with the ATCC under accession number HB 10744; or antibodies from hybridoma cell line SH4, deposited with the ATCC under accession number HB 10745.
  • the hMSCs can be distinguished from the more complex cellular microenvironment present for example in the marrow stroma ("Dexter stroma"). MSCs are distinct in morphology from Dexter stroma and also lack surface markers for T and B lymphocytes, macrophages and endothelial cells.
  • the culture conditions such as temperature pH and the like, are those previously used with hematopoietic progenitor cells, and will be apparent to the ordinarily skilled artisan.
  • the transformed hematopoietic progenitor cells are maintained in a medium that stimulates culture expansion but does not stimulate their differentiation.
  • the cells may be co-cultured in a medium such as supplemented BGJ b medium or supplemented F-12 Nutrient Mixture.
  • a medium such as supplemented BGJ b medium or supplemented F-12 Nutrient Mixture.
  • the medium is a human MSC medium which comprises Dulbecco's Modified Eagles Medium Low Glucose (DMEM-LG) (Life Technologies) supplemented preferably with fetal bovine serum.
  • DMEM-LG Dulbecco's Modified Eagles Medium Low Glucose
  • the composition can also be supplemented with an antibiotic and antimycotic composition.
  • the transduction may be effected in the absence of exogenous cytokines.
  • the hematopoietic stem cells may be transduced in the presence of one or more cytokines.
  • cytokines include interleukin-1 (IL-1), IL-6, IL-3, stem cell factor (SCF), Flt-3 ligand, leukemia inhibitory factor (LIF), granulocyte-macrophage colony stimulating factor (GM-CSF), c-kit ligand and IL-3-GM-CSF fusion protein.
  • the mesenchymal stem cells may be irradiated prior to use in order to minimize or eliminate transduction of the mesenchymal stem cells.
  • the transduced hematopoietic progenitor cells may be used alone or together with transduced mesenchymal stem cells.
  • the mixture of hematopoietic and mesenchymal stem cells may be separated to obtain a population of cells largely consisting of the transduced hematopoietic stem cells. This may be accomplished by positive and/or negative selection of transduced hematopoietic cells using antibodies to identify hematopoietic cell surface markers or by culturing out adherent mesenchymal stem cells and recovering a substantially homogenous population of transduced hematopoietic cells from the supernatant.
  • the hematopoietic stem cells may be genetically modified (transduced or transformed or transfected) by incorporation of genetic material into the cells, for example using recombinant expression vectors.
  • recombinant expression vector refers to a transcriptional unit comprising an assembly of (1) a genetic element or elements having a regulatory role in gene expression, for example, promoters or enhancers, (2) a structural or coding sequence which is transcribed into mRNA and translated into protein, and (3) appropriate transcription initiation and termination sequences.
  • Structural units intended for use in eukaryotic expression systems preferably include a leader sequence enabling extracellular secretion of translated protein by a host cell.
  • recombinant protein may include an N-terminal methionine residue. This residue may or may not be subsequently cleaved from the expressed recombinant protein to provide a final product.
  • the human hematopoietic progenitor cells transduced in the presence of human mesenchymal stem cells thus may have stably integrated a recombinant transcriptional unit into chromosomal DNA or carry the recombinant transcriptional unit as a component of a resident plasmid.
  • Cells may be engineered with a polynucleotide (DNA or RNA) encoding a polypeptide ex vivo , for example.
  • Cells may be engineered by procedures known in the art by use of a retroviral particle containing RNA encoding a polypeptide.
  • Retroviruses from which the retroviral plasmid vectors hereinabove mentioned may be derived include, but are not limited to, Moloney Murine Leukemia Virus, spleen necrosis virus, retroviruses such as Rous Sarcoma Virus, Harvey Sarcoma Virus, avian leukosis virus, gibbon ape leukemia virus, human immunodeficiency virus, adenovirus, Myeloproliferative Sarcoma Virus, and mammary tumor virus.
  • the retroviral plasmid vector is derived from Moloney Murine Leukemia Virus.
  • retroviral mediated gene transfer see McLachlin et al.(1990).
  • Suitable promoters which may be employed include, but are not limited to, adenoviral promoters, such as the adenoviral major late promoter; the cytomegalovirus (CMV) promoter; the respiratory syncytial virus (RSV) promoter; inducible promoters, such as the MMT promoter, the metallothionein promoter; heat shock promoters; the albumin promoter; the ApoAI promoter; human globin promoters; viral thymidine kinase promoters, such as the Herpes Simplex thymidine kinase promoter; retroviral LTRs; ITRs; the ⁇ -actin promoter; and human growth hormone promoters.
  • adenoviral promoters such as the adenoviral major late promoter
  • CMV cytomegalovirus
  • RSV respiratory syncytial virus
  • inducible promoters such as the MMT promoter, the metallothionein promoter
  • the promoter also may be the native promoter that controls the gene encoding the polypeptide.
  • These vectors also make it possible to regulate the production of the polypeptide by the engineered progenitor cells. The selection of a suitable promoter will be apparent to those skilled in the art.
  • the producer cell line generates infectious retroviral vector particles which include the nucleic acid sequence(s) encoding the polypeptides. Such retroviral vector particles then may be employed to transduce the hematopoietic stem cells, either in vitro or in vivo . The transduced hematopoietic stem cells will express the nucleic acid sequence(s) encoding the polypeptide.
  • telomeres can be used for this purpose.
  • Other methods can also be used for introducing cloned eukaryotic DNAs into cultured mammalian cells, for example, the genetic material to be transferred to stem cells may be in the form of viral nucleic acids.
  • the expression vectors may contain one or more selectable marker genes to provide a phenotypic trait for selection of transformed cells such as dihydrofolate reductase or neomycin resistance.
  • the hematopoietic progenitor cells may be transfected through other means known in the art. Such means include, but are not limited to transfection mediated by calcium phosphate or DEAE-dextran; transfection mediated by the polycation Polybrene (Kawai and Nishizawa 1984; Chaney et al. 1986); protoplast fusion (Robert de Saint Vincent et al. 1981). (Schaffner 1980; Rassoulzadegan et al. 1982); electroporation (Neumann et al. 1982; Zimmermann 1982) (Boggs et al. 1986); liposomes (see, e.g.
  • the present invention further makes it possible to genetically engineer human hematopoietic progenitor cells in such a manner that they produce, in vitro, polypeptides, hormones and proteins not normally produced in human hematopoietic progenitor cells in biologically significant amounts or produced in small amounts but in situations in which overproduction would lead to a therapeutic benefit. These products would then be secreted into the surrounding media or purified from the cells.
  • the human hematopoietic progenitor cells formed in this way can serve as continuous short term or long term production systems of the expressed substance.
  • This technology may be used to produce additional copies of essential genes to allow augmented expression of certain gene products in vivo .
  • genes can be, for example, hormones, matrix proteins, cell membrane proteins, cytokines, adhesion molecules, detoxification enzymes, "rebuilding" proteins important in tissue repair and drug resistance.
  • the expression of the exogenous genetic material in vivo is often referred to as "gene therapy.”
  • Disease states and procedures for which such treatments have application include genetic disorders and diseases of the blood and immune system.
  • Cell delivery of the transformed cells may be effected using various methods and includes infusion and direct depot injection into periosteal, bone marrow and subcutaneous sites.
  • the transduced cells may be used for in vitro production of desired protein(s).
  • hMSCs Human mesenchymal stem cells
  • Washed cells were resuspended in PBS to a final density of 4 x 10 7 cells/cm 2 , and a 5 ml aliquot was layered over a 1.073 g/ml Percoll solution (Pharmacia, Piscataway, NJ) and centrifuged at 900 x g for 30 minutes.
  • Mononuclear cells collecting at the interface were recovered, resuspended in human MSC medium, and plated at a density of 3 x 10 7 cells per 185 cm 2 Nunclon Solo flask (Nunc Inc., Naperville, IL). Homogeneity of the culture was determined by uniform expression of the hMSC specific surface protiens SH2, 3, and 4.
  • Human MSC medium consisted of Dulbecco's modified Eagles Medium-Low Glucose (DMEM-LG) (Life Technologies) supplemented with 10% fetal bovine serum (FBS) (Biocell Laboratories, Collinso Dominquez, CA) which was screened for its ability to support culture expansion of MSCs, and 1% antibiotic-antimycotic solution (Life Technologies).
  • FBS fetal bovine serum
  • Mesenchymal stem cell cultures were maintained at 37° C in 5% CO 2 in air, with medium changes after 48 hours and every 3-4 days thereafter.
  • the cells were recovered by the addition of a solution containing 0.25% trypsin-EDTA (Life Technologies) and replated at a density of 1x10 6 cells per 185 cm 2 flask as passage 1 cells.
  • Dexter stroma was obtained by mixing bone marrow with an equal volume of phosphate buffered saline (PBS) containing 2% bovine serum albumin (BSA) (Life Technologies), 0.6% sodium citrate (Sigma, St. Louis, MO), and 1% penicillin-streptomycin (Life Technologies), and aliquots were layered over Ficoll-Paque (1.077 gm/ml) (Pharmacia) and centrifuged at 800 x g for 20 minutes.
  • PBS phosphate buffered saline
  • BSA bovine serum albumin
  • Pharmacia 1% penicillin-streptomycin
  • the mononuclear cells that collected at the interface were suspended in medium consisting of MyeloCult H5100 (Stem Cell Technologies, Vancouver, BC, Canada) supplemented with 1 ⁇ M hydrocortisone (Sigma), plated at a density of 60 x 10 6 cells per 185 cm 2 flask, and incubated at 33°C in 5% CO 2 in air with media changes after 10 days and then every 7 days thereafter.
  • MyeloCult H5100 is made up of 12.5% FBS, 12.5% horse serum, 0.2 mM i-inositol, 20 mM folic acid, 0.1 mM 2-mercaptoethanol, 2 mM L-glutamine in Alpha MEM.
  • Primary cultures were recovered by trypsinization when the cells reached 90% of confluence and replated at a density of 1x10 6 cells per 185 cm 2 flask as passage 1 cells.
  • Peripheral blood mononuclear cells were obtained by apheresis from normal donors treated with G-CSF under an Institutional Review Board approved protocol.
  • CD34+ cells were isolated by positive selection using the VarioMacs column (Miltenyi Biotec, Inc. Auburn, CA) with a purity of 72% ⁇ 17.
  • Flasks containing fibronectin fragment CH-296 (FN) (Takara Biochemicals) were prepared according to the method described by Hanenberg et al . 1997 with modifications.
  • Bacterial ( non-tissue culture-treated) T75 cm 2 flasks (Falcon, Franklin Lakes, NJ) were coated with 17 ⁇ g/cm 2 of the recombinant Escherichia coli-derived FN fragments CH-296 (Takara Shuzo, Otsu, Japan) similarly as described (Hanenberg et al. 1996).
  • CD34+ cells were transduced with MFG- ⁇ MGMT high titer retroviral supernatant obtained as described in Reese et al . 1996.
  • ⁇ MGMT encodes a mutant form of O- 6 alkylguanine DNA alkyltransferase ( ⁇ AGT) and contains a point mutation which renders the protein resistant to the inhibitor O 6 -benzylguanine (BG).
  • BG O 6 alkylguanine DNA alkyltransferase
  • BG O 6 -benzylguanine
  • CD34+ cells transduced with ⁇ MGMT are resistant to the drug combinations BG and 1,3 bis (2-chloroethly)-1-nitrosourea (BCNU) ( Reese et al. 1996) and BG and temozolomide (TMZ) ( Reese et al. 1998).
  • BCNU 1,3 bis (2-chloroethly)-1-nitrosourea
  • TMZ
  • the CD34+ cells were transduced as follows: pMFG-wtMGMT or pMFG- ⁇ MGMT and pSV2neo DNA were cotransfected into the packaging line GP + E86, followed by viral supernatant infection of the amphotropic cell line GP + en ⁇ Am12 (Markowitz, D. et al. 1988) (Arthur Bank, Columbia University). To increase titer, a supernatant "ping-pong" method was used (Bodine et al. 1990).
  • Titer was estimated from supernatants collected after six daily media changes (Allay 1995) by infecting 1 x 10 5 K562 cells (as described below) with limiting dilutions of viral supernatant.
  • the human chronic myelogenous leukemia cell line K562 was retrovirally transduced as described by Allay 1995. Briefly, wtMGMT and ⁇ MGMT producers were treated with 10 ⁇ g/ml mitomycin C and replated.
  • K562 cells were added in the presence of human interleukin 3 (IL-3) (100 units/ml), human granulocyte-macrophage colony-stimulating factor (GM-CSF) (200 units/ml), and polybrene (8 ⁇ g/ml) and were collected after a 48-hour co-culture.
  • IL-3 human interleukin 3
  • GM-CSF human granulocyte-macrophage colony-stimulating factor
  • polybrene 8 ⁇ g/ml
  • CFU colony-forming unit
  • GM CFU-granulocyte-macrophage
  • BFU-E erythroid
  • CFU-GEMM CFU-granulocyte erythroid macrophage megakaryocyte
  • CD34+ cells (1-5 x10 5 cells/ml) were transduced in the presence of human mesenchymal stem cells (allogeneic or autologous), Dexter stroma or fibronectin fragment CH-296 (FN), described above. Control plates were coated with BSA only. Cultures were supplemented with protamine sulfate (4 ⁇ g/ml), IL-3, IL-6 (both @ 20ng/ml; Systemix, Palo Alto, CA), Flt-3 ligand (100ng/ml; Immunex) and SCF (100ng/ml; Amgen) or LIF (100 ng/ml; Systemix).
  • Transduction efficiency PCR was used to detect proviral specific sequences. To obtain genomic DNA for PCR analysis, individual colonies were resuspended in 30ul of H 2 O and boiled for 6 minutes. One ul of proteinase K solution 10 mg/ml was added and the colonies were incubated at 55°C for 2 hours and boiled again for 6 minutes. Six ul of each preparation was used per PCR reaction. Using sense primer 5'-TGGTACCTCACCCTTACCGAGTC-3' containing sequences of the MFG proviral backbone, and the antisense primer 5'-ACACCTGTCTGGTGAACGACTCT-3' specific to human MGMT ( Reese et al. 1996), transduction efficiency was determined.
  • Clonogenic efficiency The mean clonogenic efficiency of total mononuclear cells at the end of the culture was assayed as described in Allay et al. 1996.
  • vM5MGMT-transduced CD34 + cells (3 x 10 5 ) were mixed with methylcellulose (Stem Cell Technologies, Vancouver, British Columbia, Canada), 100 ng/ml stem cell factor (Amgen, Thousand Oaks, CA), 100 units/ml IL-3 (Systemix, Palo Alto, CA), 2 units/ml erythropoietin (Amgen, Thousand Oaks, CA), 100 units/ml GM-CSF (Immunex, Seattle, WA) and 0.1 mM hemin (Sigma, St.
  • FACS analysis for AGT expression ⁇ AGT was analyzed by flow cytometry as described in Liu et al. 1998, with modifications. Briefly, after cells were stabilized for 30 minutes using 1% paraformaldehyde, the membranes were permeabilized by incubating in 1% Tween 20 for 30 minutes at 37°C, (and omitted 30 minute incubation in 2% BSA/PBS). Nonspecific binding sites were blocked for 30 minutes at 22°C with 10% normal goat serum. Human AGT antibody mT3.1 (7 ⁇ g/ml) was added, and cells were incubated at 4°C overnight.
  • ⁇ AGT Light scatter was used for gating on permeabilized cells.
  • IC50 is the determination of CFU survival after exposure to TMZ or BCNU in the presence of the AGT inhibitor BG.
  • BG AGT inhibitor
  • CFU CFU-GM, BFU-E and CFU-GEMM
  • Individual CFU were collected from 10 independent experiments, each from a different donor.

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Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5061620A (en) 1990-03-30 1991-10-29 Systemix, Inc. Human hematopoietic stem cell
WO1992022584A1 (fr) 1991-06-18 1992-12-23 Caplan Arnold I Anticorps monoclonaux specifiques contre les cellules mesenchymateuses derivees de la moelle
US5197985A (en) 1990-11-16 1993-03-30 Caplan Arnold I Method for enhancing the implantation and differentiation of marrow-derived mesenchymal cells
US5226914A (en) 1990-11-16 1993-07-13 Caplan Arnold I Method for treating connective tissue disorders
US5591625A (en) * 1993-11-24 1997-01-07 Case Western Reserve University Transduced mesenchymal stem cells
WO1998020731A1 (fr) * 1996-11-15 1998-05-22 Osiris Therapeutics, Inc. Compositions mixtes de cellules msc et de precurseurs de megacaryocytes et procede pour isoler les cellules msc associees aux megacaryocytes, en separant les megacaryocytes
WO1999061588A1 (fr) * 1998-05-22 1999-12-02 Osiris Therapeutics, Inc. Production de megacaryocytes par co-culture de cellules souches mesenchymateuses humaines au moyen de cellules cd34+

Family Cites Families (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5486359A (en) * 1990-11-16 1996-01-23 Osiris Therapeutics, Inc. Human mesenchymal stem cells
AU680406B2 (en) * 1992-03-04 1997-07-31 Systemix, Inc. Culturing of hematopoietic stem cells and their genetic engineering
WO1996009400A1 (fr) * 1994-09-19 1996-03-28 Systemix, Inc. Procedes permettant de modifier genetiquement des cellules souches hematopoietiques
US7056738B2 (en) * 2001-03-23 2006-06-06 Tulane University Early stage multipotential stem cells in colonies of bone marrow stromal cells
US7592174B2 (en) * 2002-05-31 2009-09-22 The Board Of Trustees Of The Leland Stanford Junior University Isolation of mesenchymal stem cells

Patent Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5061620A (en) 1990-03-30 1991-10-29 Systemix, Inc. Human hematopoietic stem cell
US5197985A (en) 1990-11-16 1993-03-30 Caplan Arnold I Method for enhancing the implantation and differentiation of marrow-derived mesenchymal cells
US5226914A (en) 1990-11-16 1993-07-13 Caplan Arnold I Method for treating connective tissue disorders
WO1992022584A1 (fr) 1991-06-18 1992-12-23 Caplan Arnold I Anticorps monoclonaux specifiques contre les cellules mesenchymateuses derivees de la moelle
EP0592521A1 (fr) * 1991-06-18 1994-04-20 CAPLAN, Arnold I. Anticorps monoclonaux specifiques contre les cellules mesenchymateuses derivees de la moelle
US5591625A (en) * 1993-11-24 1997-01-07 Case Western Reserve University Transduced mesenchymal stem cells
WO1998020731A1 (fr) * 1996-11-15 1998-05-22 Osiris Therapeutics, Inc. Compositions mixtes de cellules msc et de precurseurs de megacaryocytes et procede pour isoler les cellules msc associees aux megacaryocytes, en separant les megacaryocytes
WO1999061588A1 (fr) * 1998-05-22 1999-12-02 Osiris Therapeutics, Inc. Production de megacaryocytes par co-culture de cellules souches mesenchymateuses humaines au moyen de cellules cd34+

Non-Patent Citations (16)

* Cited by examiner, † Cited by third party
Title
ALLAY, J. ET AL., BLOOD, vol. 85, 1995, pages 3342 - 3351
ALLAY, J.; O. KOG ET AL.: "Retroviral mediated gene transduction of human alkyltransferase complementary cDNA confers nitrosourea resistance to human hematopoietic progenitors", CLINICAL CANCER RESEARCH, vol. 2, 1996, pages 1353 - 1359
BODINE, D.M. ET AL., PROC. NATL. ACAD. SCI. USA, vol. 87, 1990, pages 3738 - 3742
EDGINGTON, BIOTECHNOLOGY, vol. 10, 1992, pages 1099 - 1106
HANENBERG, H.K. HASHINO ET AL.: "Optimization of fibronectin assisted retroviral gene transfer into human CD34+ hematopoietic cells", HUMAN GENE THERAPY, vol. 8, 1997, pages 2193 - 2206
HAYNESWORTH ET AL., BONE, vol. 13, no. 1, 1992, pages 81 - 88
LENNON D P ET AL: "HUMAN AND ANIMAL MESENCHYMAL PROGENITOR CELLS FROM BONE MARROW: IDENTIFICATION OF SERUM FOR OPTIMAL SELECTION AND PROLIFERATION", IN VITRO CELLULAR & DEVELOPMENTAL BIOLOGY. ANIMAL, TISSUE CULTURE ASSOCIATION, COLUMBIA, MD, vol. 32, no. 10, 1 November 1996 (1996-11-01), pages 602 - 611, XP008041753, ISSN: 1071-2690 *
MARKOWITZ, D. ET AL., J. VIROL., vol. 167, 1988, pages 400 - 406
MCLACHLIN ET AL., PROGRESS IN NUCLEIC ACID RESEARCH AND MOLECULAR BIOLOGY, vol. 38, 1990, pages 91 - 135
MOORE K. ET AL.: "Stromal support enhances cell- free retroviral vector transduction of human bone marrow long-term culture initiating cells", BLOOD, vol. 79, 1992, pages 1393 - 1399
NOLTA J ET AL: "Analysis of optimal conditions for retroviral-mediated transduction of primitive human hematopoietic cells", BLOOD, vol. 86, no. 1, 1 June 1995 (1995-06-01), pages 101 - 110, XP002115180 *
NOLTA J.; SMOGORZEWSKA E.; KOHN D.: "Analysis of optimal conditions for retroviral- mediated transduction of primitive human hematopoietic cells", BLOOD, vol. 86, 1995, pages 101 - 110
PITTENGER M F ET AL: "MULTILINEAGE POTENTIAL OF ADULT HUMAN MESENCHYMAL STEM CELLS", SCIENCE, AMERICAN ASSOCIATION FOR THE ADVANCEMENT OF SCIENCE, US, WASHINGTON, DC, vol. 284, no. 5411, 2 April 1999 (1999-04-02), pages 143 - 147, XP000867221, ISSN: 0036-8075 *
REESE J 0 KOÇ ET AL.: "Retroviral transduction of mutant methylguanine DNA methyltransferase gene into human CD34 cells confers resistance to 06-benzylgaunine plus 1,3-bis(2-chlorethyl)-1-nitrosurea", PROC NATL ACAD SCI USA, vol. 93, 1996, pages 14088 - 14093
REESE J S ET AL: "Human mesenchymal stem cells provide stromal support for efficient CD34+ transduction.", JOURNAL OF HEMATOTHERAPY & STEM CELL RESEARCH OCT 1999, vol. 8, no. 5, October 1999 (1999-10-01), pages 515 - 523, XP008099760, ISSN: 1525-8165 *
WELLS S ET AL: "THE PRESENCE OF AN AUTOLOGOUS MARROW STROMAL CELL LAYER INCREASES GLUCOCEREBROSIDASE GENE TRANSDUCTION OF LONG-TERM CULTURE INITIATING CELLS (LTCICS) FROM THE BONE MARROW OF A PATIENT WITH GAUCHER DISEASE", GENE THERAPY, MACMILLAN PRESS LTD., BASINGSTOKE, GB, vol. 2, no. 8, 1 January 1995 (1995-01-01), pages 512 - 520, XP008026297, ISSN: 0969-7128 *

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